Fraction collector for composition analysis

ABSTRACT

A method and apparatus for controlling fraction collection in an eluent stream flowing from an LC column. A triggering detector recognizes the presence of a target substance according to characteristics of chromatographic peaks in the eluent stream and initiates a delay timer to trigger the fraction collector. A waste stream detector is disposed at any distance from the fraction collector to detect peaks in the waste stream flowing from a fraction collector. The signature of fraction collector actuation is seen by the waste stream detector, permitting the delay time to be adjusted for optimal collection of the target compound. The presence or absence of a peak or the characteristics of a remnant peak detected by the waste stream detector are used to confirm that the target component of the eluent stream was collected as intended by the fraction collector.

REFERENCE TO RELATED APPLICATIONS

This patent application is a Divisional of application Ser. No.10/427,324, filed May 1, 2003 contents of which is incorporated hereinby reference in its entirety and claims the benefit thereof.

FIELD OF THE INVENTION

This invention relates to a fluid system and particularly to fractioncollection in a liquid chromatography (“LC”) eluent stream.

BACKGROUND OF THE INVENTION

Liquid chromatography is commonly used for analyzing the composition ofchemical compounds whereby solutions containing the compounds to beanalyzed are forced under pressure through a liquid chromatography (LC)column. The LC column is specifically constructed to interact with thepressurized solution and affect the fluid flow rate in a way that ischaracteristic of the composition of chemicals in the solution.Components are thereby separated in the solution according to theirchemical composition and corresponding flow rate. Eluent from the LCcolumn is typically in a highly diluted liquid phase. Separatedcomponents are manifested as “chromatographic peaks” on detectorapparatus.

In liquid chromatography analysis, the choice of an appropriateseparation strategy, including the combined implementation of hardware,software, and chemistry, results in the separation of an injected sampleinto separate components, which elute from the column in reasonablydistinct zones or “bands”. As these bands pass through a detector, adetector output, usually in the form of an electrical signal, isproduced. The pattern of analyte concentration within the eluting bands,which can be represented by means of a time-varying electrical signal,gives rise to the nomenclature “chromatographic peak” (or “peak”). Peaksmay be characterized with respect to their “retention time,” which isthe time at which the center of the band transits a detector. In manyapplications, the retention time of a peak is used to infer the identityof the eluting analyte based upon related analyses with standards andcalibrants. The retention time for a peak is strongly influenced by themobile phase composition of the analyte and by the accumulated volume ofmobile phase which has passed through the LC column.

It is often desirable to separate and collect specific component(s) fromthe HPLC separation of a complex mixture for further testing andevaluation. For example, samples of a pure component may be needed toevaluate the biological activity or other properties of a candidate drugmolecule.

Conventional methods for physically collecting a purified sample from aneluent stream employ a fraction collector which is capable of divertinga portion or “fraction” of the stream into a collection vessel at aspecified time. The specified time for opening the fraction collectorideally coincides with the arrival at the fraction collector ofconcentrated components of the eluent stream.

As is known in the art, timing of a fraction collector in an LC eluentstream can be controlled by installing a triggering detector whichinitiates a timer upon detection of a particular component concentration(peak). The fraction collector is triggered after a delay time elapses.

Any detector which senses a peak before it reaches the fractioncollector can be used to trigger fraction collection. UV detectors arecommonly used as triggering detectors in UV-directed purification orfraction collection systems. Mass spectrometers are commonly used astriggering detectors in mass-directed purification or fractioncollection systems. These detectors can recognize a signature peakrepresenting the presence of particular components and trigger thefraction collector to collect only desired components from the eluentstream. The triggering detector can be installed upstream of a fractioncollector if it is a non-destructive detector such as a UV detector.However, the triggering detector is not necessarily installed directlyupstream of a fraction collector. It can also be installed in one branchof a split flow wherein the other branch of the split flow is directedto the fraction collector. For example, if a destructive detector suchas a mass spectrometer is used as a triggering detector, it must beinstalled in a separate branch of the eluent stream.

The delay time is the time between the appearance of a peak at thetriggering detector and the arrival of the peak at the fractioncollector. This time depends on the flow rate and fluid volume in therelevant connecting tubing. The delay time of an eluent stream can bedetermined by injecting a known calibrant such as a visible dye at anupstream location and recording the elapsed time between its detectionby the triggering detector and its arrival at the fraction collector.One drawback of this calibration method is that the special calibrantdye must be injected whenever it is desired to check that the correctdelay time is being used. For example, a change in flow rate for anyreason will result in a change in delay time, which could go unnoticed.

Mass spectrometers are commonly used to analyze the composition of aneluent stream. U.S. Pat. No. 6,406,633 to Fischer et al. (“Fischer'633”) and U.S. Pat. No. 6,106,710 to Fischer et al. (“Fischer '710”)disclose a fraction collection system which directs a portion of aneluent stream from a liquid chromatography column to a massspectrometer, and detects a desired component (peak) with the massspectrometer. The apparatus of the Fischer disclosures is showndiagrammatically in FIG. 1. The mass spectrometer 52 triggers a delaytimer for controlling the actuation of a fraction collector 54 in aseparate branch of the stream. The eluent stream flows from a liquidchromatography column 51 to a splitter 57 which divides the eluentstream and directs one branch to a mass spectrometer 52 and anotherbranch to a fraction collector 54. Even though the mass spectrometer 52disclosed in Fischer '633 and Fischer '710 is disposed in a separatestream branch 53 rather than directly upstream of the fraction collector54, the mass spectrometer 54 can be used to time the opening of thefraction collector 54 if the flow rates in each branch of the stream arerelated in a predictable way, and the peak is detected by the massspectrometer before it reaches the fraction collector. A downstreamdetector 55 is disposed near the fraction collector 54. Detection of apeak at the downstream detector 55 allows the flow rate of the peak tobe determined by measuring the elapsed time between detection of asample upstream and its arrival at a downstream point near the fractioncollector. The downstream detector 55 described in Fischer '633 andFischer '710 is a non-destructive detector such as a UV detector.

To effect timing of the fraction collector 54 Fischer '633 discloses adelay time determined empirically by the injection of a calibrant in theeluent stream and timing the arrival of the calibrant at the massspectrometer 52 in one branch of the stream. The arrival of thecalibrant at downstream detector 55 proximate to fraction collector 54in a separate branch of the stream is also timed. The delay betweenarrival of the calibrant at the mass spectrometer 52 and arrival of thecalibrant at the downstream detector 55 provides flow rate informationthat can be used to time the opening of the fraction collector after asample being analyzed is detected in the mass spectrometer.

Each of the various implementations described in Fischer '633 andFischer '710 involves the use of a destructive detector such as a massspectrometer to identify the presence of a substance in an eluentstream. The use of a destructive detector requires splitting the eluentstream into an analyzed stream flowing to the destructive detector and acollection stream flowing to the fraction collector. Fischer '633 andFischer '710 also disclose various implementations of a third detectorupstream from the fraction collector to better characterize the flow ineach branch of the eluent stream in order to more accurately time theopening and closing of the fraction collector. The third detector isdescribed in Fischer '633 and Fischer '710 as a non-destructive detectorsuch as a UV detector.

The various implementations described in Fischer '633 and Fischer '710require placement of a non-destructive detector near the fractioncollector. Such placement is disadvantageous for use because typicalfraction collectors use long robotic arms to dispense into collectionvessels. If a detector must be located near a fraction collectordispensing head, only detectors suitable for mounting on a robotic armcan be used. This precludes use of standard HPLC detectors, for examplea tunable UV detector, which can be used to detect a variety ofcomponents being separated, but are not suitable for mounting on arobotic arm.

No information regarding the accuracy of the fraction collector timingsignal is provided by any apparatus described in Fischer '633 or Fischer'710. Furthermore, the various apparatus described in Fischer '633 andFischer '710 do not provide confirmation that a desired component wassuccessfully collected by a fraction collector. The delay time in Fisher'633 is determined using a calibrant. It is assumed to remain unchangedduring sample collection, until such time as calibration is repeated.

Each of the various fraction collection methods described in Fischer'633 requires the use of a calibrant which is injected into the eluentstream for calibrating the delay period before the fraction collector isactuated. Persons skilled in the art will recognize that any calibrantmay have different flow characteristics than the substance beinganalyzed. For example, a change in flow rate or split ratio could occurafter calibration is performed. The calibrated delay period thereforemay include errors that result in non-optimal collection of a desiredsubstance at the fraction collector. Even small errors in timing cancause a fraction collector to miss much or all of a target substance.

None of the methods and apparatus heretofore known for controllingfraction collector timing in LC systems include a means to confirm thesuccessful collection of a desired component in the eluent stream.

SUMMARY OF THE INVENTION

The present invention provides a method and apparatus for controllingfraction collection in an eluent stream flowing from an LC column. Atriggering detector recognizes the presence of a target substance abovea predetermined threshold level according to characteristics ofchromatographic peaks in the eluent stream. A waste stream detector isdisposed to detect peaks in the waste stream flowing from a fractioncollector. Presence or absence of a peak in the waste stream above apredetermined threshold level indicates whether the peak was properlycollected by the fraction collector. Characteristics of peaks detectedby the waste stream detector are used to confirm that the targetcomponent of the eluent stream was actually collected by the fractioncollector and to calibrate the timing of the fraction collector foroptimal collection of the target component. Timing calibration dependson recognizing a fraction collector event, opening or closing, in thewaste stream detector's signature of a peak. The timing calibration canbe automated. There is no requirement for the waste stream detector tobe placed close to the fraction collector. The waste stream can be ledto any detector located in the purification system package.

An illustrative embodiment of the invention provides a method forcollecting a sample component from an eluent stream out of a liquidchromatography (LC) column. The eluent stream is directed to a fractioncollector for diversion of a specific portion or fraction of the streamto a collection vessel or alternate path for further analysis,separation or evaluation of the fraction. A waste stream carries theremainder of the eluent stream away from the fraction collector. Thewaste stream ceases to flow while the fraction collector is actuated.Characteristics of chromatographic peaks from the target samplecomponent are detected in the waste stream. A calibrated delay time foractuating the fraction collector is computed according to thecharacteristics of peaks detected in the waste stream. The fractioncollector timing is adjusted to effect optimum fraction collection.

The target component is first detected by a triggering detector beforeit reaches the fraction collector in the eluent stream. The fractioncollector is actuated when an estimated delay time has elapsed afterdetection of a desired sample by the triggering detector. Some portionsof the sample component are detected at the waste stream detector. Thedelay time used to open the fraction collector is tuned by evaluatingthe shape of chromatographic peaks that are detected in the waste streamand adjusting the delay time in response to characteristics of thepeaks.

Non-destructive optical detectors such as UV detectors are particularlysuitable for use as waste stream detectors in the various illustrativeembodiments of the invention. The chromatograms output by UV detectorsare tuned to a wavelength appropriate for detecting a desired peak. UVdetectors are also particularly well suited for use as triggeringdetectors according to the present invention.

In another illustrative embodiment of the invention, a splitter isdisposed upstream of the fraction collector and diverts a portion of thestream to a destructive detector such as a mass spectrometer. A delaytimer for actuating the fraction collector can be initiated upon thedetection of a desired peak by the mass spectrometer. Detection of peaksby the mass spectrometer can be used in the same manner as thetriggering detector described hereinbefore if the flow path of the massspectrometer is shorter (temporarily) than the flow path of the fractioncollector so that a portion of the desired component reaches the massspectrometer before a corresponding portion of the desired componentreaches the fraction collector. Only about 1/1,000 of the flow isdirected to the mass spectrometer, and as a practical matter, a make-uppump is used to dilute the sample and speed it to the mass spectrometer.The high concentration of sample in the preparative stream is too highto inject directly into the electrospray interface of a massspectrometer.

The present invention can also be implemented using a non-destructivetriggering detector disposed upstream of a splitter. The splitterdirects a branch of the eluent stream to any type of analysis equipmentsuch as a mass spectrometer. The splitter directs a separate branch ofthe eluent stream to the fraction collector. Either the non-destructivetriggering detector or the mass spectrometer can be used to detect thepresence of a desired peak and initiate the delay timer to operate thefraction collector. Alternatively, a non-destructive detector can belocated downstream of the splitter in either the stream branch directedto the destructive detector or in the stream branch directed to thefraction collector.

Any detector can be used as the waste line detector. In place of the UVdetector discussed above, any non-destructive or destructive detectorcan be used. In particular when a mass spectrometer is employed as thetriggering detector, a portion of the waste stream can be analyzed bymultiplexing into a second analytical channel of the same massspectrometer.

In each of the illustrative embodiments, a waste stream detectorprovides information used to adjust the timing of the fractioncollector. For example, a full peak detected in the waste streamindicates that a target component was not collected by the fractioncollector. Contrarily, a peak detected in a triggering detector but notin the waste stream, indicates that the fraction was collected properlyat the fraction collector. Characteristics such as the width of a peakin the waste stream can be compared to similar characteristics of anupstream peak. A substantial match of such characteristics indicatesthat substantially the entire volume of a peak was missed by thefraction collector. A partial match between such characteristicsindicates that only a portion of the peak was collected by the fractioncollector. The particular differences between the shape of the peak atthe waste stream detector with and without the fraction collectoropening can be used to determine a precise actuation time adjustmentthat enables collection of the optimal volume of a target sample by thefraction collector.

In at least one illustrative embodiment, the delay period between theupstream detection of a target sample and the actuation of a fractioncollector is adjusted during a setup operation. The fraction collectortiming is verified and if necessary adjusted according tocharacteristics of peaks detected in the waste stream, periodically oreach time the fraction collector is actuated.

In another illustrative embodiment, a calibrant component havingproperties detectable by both the triggering detector and the wastestream detector is injected upstream of the triggering detector during asetup process. The fraction collector can be timed to partially collectthe calibrant peak. Characteristics of calibrant peaks detected in thewaste stream can be used to calibrate the timing of the fractioncollector.

The present invention overcomes the disadvantages of previously knownmethods by confirming the collection of a target sample in an eluentstream of a liquid chromatography (LC) column. The present inventionfurther overcomes disadvantages of the prior art by optimizing thetiming of a fraction collector to minimize the loss of target samplecomponents. Timing information determined using data from the wastestream detector in the present invention allows continuous recalibrationof the fraction collector timing.

The waste stream detector according to the present invention is notnecessarily installed near the fraction collector or on the fractioncollector dispensing arm, which makes large and rapid motions. Rather,the waste stream detector can be installed remotely from the fractioncollector dispensing arm. This allows standard HPLC detectors to be usedwhich operate on any separated peak, and do not require the prior artrecourse to a calibrant dye. In addition, such placement overcomes manydisadvantages of the prior art by facilitating use of parallel fractioncollector dispensing heads with a single multiplexed waste streamdetector. Such placement also allows the use of a standard UV detectorhaving tunable wavelengths to detect a variety of compounds beingseparated. Placement of a detector in the waste stream according to thepresent invention also allows the use of a single multi-channel detectorfor both triggering detector and waste stream detector.

As examples, a single UV detector with 8 parallel flowcells could beused for the triggering detectors as well as the waste line detectorsfor a 4-channel purification system. Similarly, an 8-way multiplexedmass spectrometer could be used to provide four independent triggeringchannels plus analysis of the four waste lines. In each case the delaytiming of the four independent fraction collectors can be determinedaccording to the invention.

The various embodiments of the present invention also provide advantagesover previously known fraction collector techniques by eliminating theneed to place a detector at or near the fraction collector, or to injecta calibrant component into the eluent stream. The present invention alsoeliminates fraction collector timing errors introduced by reliance inthe prior art on flow characteristics of a calibrant that may bedifferent from flow characteristics of a desired component at a latertime in the eluent stream.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments, taken in conjunction with the accompanyingdrawings in which:

FIG. 1 is a diagrammatic representation of a system flow path includinga downstream detector proximate to a fraction collector in a liquidchromatography eluent stream as known in the prior art;

FIG. 2 is a diagrammatic representation of a system flow path includinga triggering detector and a waste stream detector according to anillustrative embodiment of the present invention;

FIG. 3 is a diagrammatic representation of a system flow path includinga mass spectrometer and a waste stream detector according to anillustrative embodiment of the present invention;

FIGS. 4(a)-4(g) are timing diagrams illustrating the shape of remnantpeaks detected in the waste stream relative to the actuation timing of afraction collector;

FIG. 5 is a chromatogram of an exemplary peak detected by waste streamdetector juxtaposed to a chromatograph of the same exemplary peakdetected by a triggering detector;

FIG. 6 is a chromatogram of an exemplary peak detected in the wastestream after a fraction collector is actuated with too short a delaytime;

FIG. 7 is a chromatogram of an exemplary peak detected in the wastestream after a fraction collector was actuated with near optimal timing;

FIG. 8 is a chromatogram of an exemplary peak detected in the wastestream after a fraction collector was open with too long a delay time;and

FIG. 9 is a process flow diagram of a calibration method according to anillustrative embodiment of the invention.

DETAILED DESCRIPTION

The present invention will be described in detail with respect tochromatographic applications with the understanding that embodiments ofthe present invention are directed to industrial and process controlapplications as well.

Referring to FIG. 2, a system flow path 10 according to an illustrativeembodiment of the invention is shown diagrammatically. The system flowpath 10 includes a triggering detector 12, a fraction collector 14 and awaste stream detector 16 arranged in series. A delay timer 15 isconfigured to introduce timed signals for actuating (opening andclosing) the fraction collector 14. An eluent stream 18 from a liquidchromatography column (LC Column) (not shown) flows through thetriggering detector 12 and continues on to the fraction collector 14.The fraction collector 14 is triggered by the delay timer 15 at anappropriate time to divert and collect or identify a particularcomponent of the eluent stream 18. The portions of the eluent stream 18that are not diverted by the fraction collector 14 continue along theflow path 10 into a waste stream 20. The waste stream 20 flows throughthe waste stream detector 16. The function of delay timer 15 may beprovided by the chromatography management system software.

The eluent stream 18 includes a series of separated sample components insolution. Particular components are identifiable by the triggeringdetector 12 and the waste stream detector 16 as chromatographic peaks inthe detector response. A chromatographic peak indicating the presence ofa desired component in the eluent stream 18 at the triggering detector12 initiates the fraction collector delay timer 15. The desired peakarrives at the fraction collector after a time delay that depends on theinternal volume of tubing connecting the triggering detector to thefraction collector and on the flow rate of the component in the eluentstream. The fraction collector is opened when the time delay elapses andcloses after a second delay. The second delay can be predetermined ordetermined by touch-down of the trailing edge of the peak at thetriggering detector 12. The waste stream detector 16 detects the remnantpeak in the eluent stream. The shape and timing of the remnant peakprovide information to establish the optimal timing of fractioncollection and provide an ongoing monitor to determine whether thedesired fractions have been collected. The waste stream detectorprovides a signal which can automatically fine-tune the delay timebetween detection of a peak by the triggering detector 12 and opening ofthe fraction collector 14 to optimize the collection of componentsamples.

In the setup process described below the fraction collector 14 can betimed to begin collection before a peak reaches it and to stopcollecting before the peak has passed completely. The waste detector 16senses the portion of the peak that was not collected. Detection of asignal exceeding a predetermined threshold on the waste detectorchromatogram indicates the point on the peak where the fractioncollector stopped collecting. The optimum fraction collector delay timecan be found by comparing the trace to a reference chromatogram obtainedby the waste stream detector after allowing a peak to pass uncollectedby the fraction collector.

The setup process described here is one way the signature of the wastestream detector can be used to establish the fraction collector delaytime, t_(delay). As a first step a peak detected by the triggeringdetector at time t=0 is allowed to flow to the waste stream detectorwithout opening the fraction collector. Detection of the peak by thewaste stream detector at time t₁ establishes the closed system flowtime. From the length and internal diameter of connecting tubing,estimate the system volume between triggering detector 12 and fractioncollector 14 (V₁) and between the fraction collector and waste streamdetector 16 (V₂). Then the time for a peak to reach the fractioncollector from the triggering detector can be estimated ast _(est) =t ₁ ·V ₁/(V ₁ +V ₂)

Make a second injection with the fraction collector delay time set tot_(setup)=t_(est)−Δt_(fc)/2 where Δt_(fc) is the time that the fractioncollector remains open, set initially to the width of the eluting peakas measured by the triggering detector. Setting the fraction collectordelay time t_(delay) to t_(setup) will cause the fraction collector tostart collecting before the peak reaches it, diverting mobile phase intothe collection vial, plus the leading portion of the peak. The wastestream detector will subsequently show a response for the trailingportion of the peak not collected. The onset of this response at t₂allows t_(error) to be calculated. t_(error) is the amount by whicht_(setup) must be increased to give a fraction collector delay timet_(delay) which will collect the peaks correctly.t _(error) =Δt _(fc)−(t ₂ −t ₁)t _(delay) =t _(setup) +t _(error)

In an illustrative embodiment, the triggering detector 12 comprises atunable UV detector such as a Model 2487 or a Model 2996 photodiodearray detector available from Waters Corp., Milford, Mass. A UV detectorsuch as a Waters Model 2487 detector is suitable for use as a wastestream detector 16. In another illustrative embodiment, the triggeringdetector 12 and the waste stream detector 16 can be implemented usingtwo analytical channels of a single instrument. For example, twochannels on a parallel UV detector such as Waters Model 2488 4 or 8channel UV detector, or two channels on a Micromass MUX MassSpectrometer from Waters Corporation can be used as the triggeringdetector and waste stream detector according to the present invention.

In an illustrative embodiment of the invention, the fraction collectordelay timer 15 is a data system including a computer having dataacquisition and control functionality operatively connected to thetriggering detector, fraction collector and waste stream detector.Suitable software for use in a data system to implement the controltimer 15 according to illustrative embodiments of the invention includesEmpower software from Waters Corporation of Milford, Mass., or MassLynxsoftware available from the Micromass division of Waters Corporation.

The shape of a chromatographic peak at the waste stream detector isanalyzed to determine whether the fraction collector 14 was opened andclosed at appropriate times for collecting the desired peak. Suchanalysis of the waste stream peak in relation to the collected samplecan be implemented via mathematical functions, as will be appreciated bythose skilled in the art, modeling the operations as described ingreater detail hereinafter.

Turning now to FIG. 3, a system flow path 10 of another illustrativeembodiment is described wherein an eluent stream 18 flows from a liquidchromatography column 19 to a stream splitter 22 which directs part ofthe eluent stream to a destructive detector such as a mass spectrometer24. The remainder of the eluent stream continues to a fraction collector14 and then on to a waste stream 20 having a waste stream detector 16disposed therein. The embodiment illustrated in FIG. 3 replaces thefunction of the triggering detector 12 with the mass spectrometer 24wherein the mass spectrometer 24 triggers the fraction collector delaytimer upon recognition by the mass spectrometer of a leading edge orthreshold level of a desired chromatographic peak.

The presence of the stream splitter 22 in the embodiment of FIG. 3influences the flow rate of the eluent stream 18 downstream of thesplitter and to the mass spectrometer, thereby requiring a differentdelay time than that required for the system flow path of the embodimentillustrated in FIG. 2. Nonetheless, calibration of a fraction collectoractuation time can be implemented in the embodiment of FIG. 3 accordingto the method of the present invention as described herein wherein thedelay time is determined to compensate for the effect of additional pathsections in the system flow path 10. Flow rates are more susceptible tochange when split flows are involved. The automatic checking andfine-tuning of timing calibration made possible by the present inventionis particularly valuable.

Calibration of the fraction collector timing and confirmation of samplecollection by the fraction collector using chromatograms output from thewaste stream detector is described with reference to FIGS. 4(a)-4(g).Seven illustrative sets of chromatograms are shown corresponding toseven different controller delay conditions. The columns in FIGS. 4(a)to 4(g) represent time increments wherein a peak under consideration(diagrammed as a triangle) transits the triggering detector during thetime period represented in the first column 81; the peak underconsideration transits the fraction collector during the time periodrepresented by the second column 83; and the peak under considerationtransits the waste detector during the time period represented by thethird column 85. It should be noted that no chromatogram is actuallyacquired as the peak transits the fraction collector as indicated in thesecond column 83. Rather, the second column 83 illustrates the shape ofthe peak under consideration that would be detected by a hypotheticaloptical detector in the fraction collector as the peak transits thefraction collector.

The first controller delay condition as represented in FIG. 4(a) occurswhen the fraction collector is not triggered at all. This occurs, forexample, when an experiment is performed to establish the time t₁ for apeak to transit from the triggering detector to the waste detector whenthe fraction collector is closed. For the purpose of this discussion,all times are referenced to t=0 when the triggering detector detects theleading edge of a peak. The leading edge 80 of a peak 82 is detected atthe triggering detector and starts the delay timer at time t=0. Theleading edge 80′ of a peak 82′ arrives at the fraction collector at timet_(delay) (wherein t_(delay) is not yet established). Since no signal ispresent to open the fraction collector, the entire peak 82″ continues inthe waste stream and its leading edge 80″ is detected by the wastestream detector to establish t₁. The chromatogram for the peak 82detected by the triggering detector at t=0 is substantially the same asthe chromatogram for the peak 82″ obtained from the waste streamdetector at t₁ because no fraction is collected.

FIG. 4(b) illustrates a controller delay condition which causes thefraction collector to open and close before the peak reaches it. Amobile phase portion of the eluent stream without the desired componentsis diverted into collection vessel ahead of the peak. Therefore, thechromatogram signals 86′ and 86″ are the same as 82′ and 82″. Thechromatogram signals 86′ and 86″ do not provide delay timinginformation. A fraction collector timing signal 88 has a leading edge 89corresponding to a fraction collector open signal t_(setup) and atrailing edge 90 corresponding to a fraction collector close signal. Theperiod during which the fraction collector is open (Δt_(fc)) isrepresented by the width 92 of the timing signal 88.

FIG. 4(c) represents chromatograms resulting from a timing signal 96which occurs earlier than the optimal time for opening and closing thefraction collector but late enough to cause the fraction collector tocollect the leading part 98 of a peak 94′. This provides informationneeded to compute the appropriate delay time. The fraction collectoropens at time t_(setup) 97 which occurs before any part of the peak 94reaches the fraction collector. Mobile phase ahead of the peak and theleading part 98 of the peak 94′ is collected before the fractioncollector closes. The fraction collector closes at timet_(setup)+Δt_(fc) 100. The substantial part 94″ of the peak goes to thewaste stream.

To optimally collect the peak, the fraction collector should have openedupon arrival at the fraction collector of the leading edge threshold ofpeak 94′. The event in the waste stream detector chromatogram due to thefraction collector closing is at time t₂. Consequently, the waste streamdetector chromatogram 94″ shows that the fraction collector actuallyclosed at time t₂−t₁ 95 after arrival of the peak leading edge thresholdat the fraction collector. The error in the delay time is thereforet_(error)=Δt_(fc)−(t₂−t₁). The optimal time to trigger the fractioncollector is t_(delay)=t_(setup)+t_(error)=t_(setup)+Δt_(fc)−(t₂−t₁).

The waste stream detector signal shown in 4(d) occurs when the fractioncollector was opened at the substantially optimal time and confirms thatthe peak was actually collected. The leading edge 104 of the fractioncollector timing signal 106 substantially corresponds to the arrival ofthe leading edge 108 of the desired peak 110′ at the fraction collector.The trailing edge 112 of the fraction collector timing signal 106substantially corresponds to the passage of the trailing edge 114 of thedesired peak. The leading edge of the peak passes the fraction collectorbefore collection begins and is frozen in the waste stream as thefraction collection proceeds. When the fraction collector closes(collection is completed), the trailing remnant of the peak is pushed incontact with the leading remnant. Together they flow to the waste streamdetector. Only a small residual volume 116 of a peak is indicated on thewaste stream chromatogram because the bulk of the fraction is collected.

FIG. 4(e) represents chromatograms resulting from a timing signal 118which occurs later than the optimal time for opening and closing thefraction collector but early enough to cause the fraction collector tocollect the trailing part 120 of a peak 122′. The fraction collectoropens at time t_(setup) 124 which occurs after the leading part 126 ofthe peak 122′ has transited the fraction collector. The leading portionof the peak is frozen in the waste line ahead of the waste streamdetector. The trailing part 120 of the peak 122′ is collected and thefraction collector closes after a period Δt_(fc). After a delay ofΔt_(fc) the leading part 126′ of the peak flows to the waste streamwhere it is detected by the waste stream detector. The event in thewaste stream detector chromatogram due to the fraction collector closingis at t₂. The timing error can be calculated according to the equationt_(error)=t₂−(t₁+Δt_(fc)). The late operation of the fraction collectorcan be adjusted as described herein according to the equationt_(delay)=t_(setup)+t_(error) or equivalentlyt_(delay)=t_(setup)+Δt_(fc)−(t₂−t₁). Note that t_(error) in this case isnegative.

In the illustrative embodiments of FIGS. 4(c) and 4(e) the fractioncollector event occurs at time t₂ in the waste stream chromatogram. Whenthe fraction collector opens early, FIG. 4(c), the event is at the startof the remnant peak and when the fraction collector opens late, theevent is at the end of the remnant peak. The delay of the remnant peakby the fraction collector open time, Δt_(fc), in FIG. 4(e) distinguishesthe two cases. It should be noted that retardation and advancement ofthe fraction collector timing is measured with respect to detection of apeak in the triggering detector which is used to initiate the delaytimer to open the fraction collector.

In FIG. 4(f) it can be seen that the fraction collector timing signal130 opens the fraction collector after a peak 134′ has passed. The flowin the waste stream stops when the fraction collector is open so thepeak 134″ is “frozen” in the waste stream during that period.Accordingly, when a peak 134″ in the waste stream is delayed by a periodcorresponding to Δt_(fc), it can be inferred that the timing signal 130occurred after the entire peak 134″ had passed the fraction collector.Advancement of the timing signal is necessary but the magnitude oft_(error) can not be inferred from the waste stream chromatogram whenthe entire peak is missed by the fraction collector.

In FIG. 4(g) the fraction collector open signal T_(FC) 136 occurs aftera peak has entered the waste stream detector. Since the waste streamdoes not flow while the fraction collection is open, absorbance in thewaste stream detector is frozen for a time Δt_(fc) 140 after which theremaining part 142 of the peak passes the waste stream detector. As inFIG. 4(f) it can be inferred that the fraction collector opened toolate, and none of the peak was collected. The correct delay time can notbe determined from this chromatogram.

Although, band spreading or broadening of the peak often occurs as aneluent stream flows between the triggering detector and the waste streamdetector, the timing differences used above recorded at the waste streamdetector are not generally affected.

As can occur, the peaks in FIG. 4 may be wider than shown and peaks atthe triggering detector, fraction collector and waste stream detectoroverlap in time. This does not affect the ability to compute t_(delay)from the waste stream detector chromatogram as described above.

Actual examples of chromatograms using two UV absorbance detectors areshown in FIGS. 5-8. They illustrate the cases described with the aid ofthe diagrams in FIG. 4. A diode array detector Waters Model 2996 is usedfor the triggering detector and a Waters Model 2487 UV detector is usedfor the waste stream detector. Both detectors measured absorbance at 254nm. Note that the peak has an unresolved minor component in its tail andthe upstream peak (FIG. 5 a) has broadened significantly before reachingthe waste stream detector (FIG. 5 b). These factors do not interferewith the ability of the described method to compute fraction collectordelay time from the waste stream detector chromatogram.

The fraction collector was triggered at steadily increasing delay timesfrom the threshold detection of the triggering detector. The delay timeclock is triggered to start (t=0) as the threshold of the peak isdetected by the triggering detector. All timing information to correctthe fraction collector delay time for optimal collection then comes fromobservation of the waste stream detector signature. (Note: the timingmarks on FIGS. 5-8 start at an arbitrary point before the peak reachesthe triggering detector) FIG. 5 b shows the waste stream signature whenthe fraction collector does not open, similar to the case shown in FIG.4 a. Some band broadening is evident in the waste stream signature,which affects all waste stream chromatograms to the same extent.

An example will be described wherein the invention was embodied with thetriggering detector 12 was a UV-visible photodiode array detector byWaters Corporation, Model No. 2996. The waste stream detector 16 was adual wavelength tunable wavelength UV-visible detector by WatersCorporation, Model No. 2487. The triggering detector 12 and the wastestream detector 16 were both configured to detect peaks absorbing at awavelength of 254 nanometers. Referring now to FIG. 5, a waste streamchromatogram 102 and an upstream chromatogram 101 as detected by thetriggering detector 12 and waste stream detector 16 of the presentexample are represented. The substantial similarity between the wastestream peak 103 and the upstream peak 105 is consistent with no part ofthe peak being collected by the fraction collector, which during thisrun did not open. A similar result would have been obtained had thefraction collector opened, but too early to collect any portion of thepeak. Compare with FIGS. 5A and 5B.

FIG. 6 represents a waste stream chromatogram 216 rendered by theapparatus of the present example wherein the time delay betweendetection of a peak at the triggering detector and opening of thefraction collector is configured to 15 seconds (t_(setup)). In thepresent example, the fraction collector can be seen to have closed 0.08minutes (approximately 5 seconds) after the peak arrived at the fractioncollector. The vertical leading edge 218 in the waste stream peak 220indicates an early actuation and early closing of the fractioncollector. (See also the analogous illustration in FIG. 4(c)). Theleading portion of the peak 220 was collected by the fraction collectoras evidenced by its absence from the waste stream detector chromatogram.The timing delay error (t_(error)) is calculated as Δt_(fc) minus 5seconds. t_(error) can be added to t_(setup), the fraction collectordelay time used in FIG. 6, to calibrate the desired fraction collectortiming.

Referring now to FIG. 7 the apparatus of the example was configured toopen the fraction collector 25 seconds after a peak is detected at thetriggering detector (t_(setup)=25 sec.). Most of the peak is collectedby the fraction collector indicating substantially optimal calibration.A small peak 222 detected in the waste stream represents the trailingedge of the main peak. In some embodiments of the invention, it may bedesirable to avoid collecting the trailing edge of the main peak,because the trailing portion of a peak may contain contamination. Thefraction collector timing in this case allowed the trailing edge of thepeak to pass to the waste stream.

FIG. 8 illustrates a chromatogram 224 rendered by the waste streamdetector of the present example wherein the delay time is set to 35seconds. Here, the leading edge 226 reaches the waste stream detectorbefore the fraction collector is opened. (See also, the analogousillustration in FIG. 4(g)). UV absorbance by the waste stream detectoris frozen during the time that the fraction collector is open (Δt_(fc))225. The remainder 227 of the peak 223 then flows into the waste streamwhere it is detected by the waste stream detector.

Referring now to FIG. 9, a system flow diagram illustrates the method ofcalibrating a fraction collector according to an illustrative embodimentof the invention. A first set-up run is performed 230 wherein a peak isinjected upstream of the triggering detector. The peak is allowed toflow from the triggering detector to the waste stream detector withouttriggering the fraction collector. Times of detection of the peakthreshold by the triggering detector t=0 and the waste stream detectort₁ is noted. This provides information about the flow time between thetriggering detector and the waste stream detector. A second set-up runis then performed 232 wherein the fraction collector is intentionallytriggered early or late at time t_(setup) such that the fraction closingor opening event is observed in the waste stream detector signature. Thetime of this event in the waste stream signature is t₂. This combinedwith t₁ provides information from which to calculate t_(error) 234.Finally t_(error) is added 236 to t_(setup) to compute t_(delay) tocalibrate the fraction collector and thereby facilitate collection ofsubstantially all of the peak. (See also FIG. 4 d). The value oft_(error) is positive when the fraction collector opens early, butnegative when the fraction collector opens late.

Although the illustrative embodiments are described herein primarilywith respect to a fraction collector timing control signal calibratedwith reference to output from a UV type waste stream detector incombination with an upstream UV detector to provide peak identificationand triggering, persons having ordinary skill in the art shouldappreciate that various optical detectors such as refractive indexdetectors or fluorescence detectors can be used to detect peaks in thewaste stream (or upstream) and provide calibration information accordingto the method and apparatus of the present invention. Persons skilled inthe art should appreciate that destructive detectors such as massspectrometers, evaporative light scattering detectors or nitrogendetectors can also be used to detect peaks in the waste stream (orupstream using a splitter) and provide calibration information forfraction timing according to the method and apparatus of the presentinvention. Mixed detectors are also feasible, such as a massspectrometer to identify the fraction upstream and start the delaytimer, in combination with a UV detector in the waste stream.

Although embodiments of the present invention are described with respectto eluent streams from a liquid chromatography column, persons havingordinary skill in the art should appreciate that the method of thepresent invention can be used to calibrate timed collection devices invarious different fluid analysis systems.

Although embodiments of the invention are described herein which measuretiming signals according to the detection of a threshold, or lift-offportion of a detected peak, persons having ordinary skill in the artshould appreciate that other portions of the peak, such as the top pointof a detected peak, can also be used to measure timing signals andperform fraction collector calibration according to the invention.

Although the invention is described herein in terms of detecting asingle component in an eluent stream, persons skilled in the art shouldappreciate that the apparatus of the present invention could also beprogrammed to recognize peak characteristics of multiple components inan eluent stream and trigger the fraction collector to collect themultiple components.

Although the invention is described herein in terms of calibrating thetiming of a fraction collector and confirming the collection ofcomponents, persons skilled in the art should appreciate thatcharacteristics of signals detected by a waste stream detector accordingto the invention can be used for a number of other purposes. Forexample, various qualities of collected samples can be inferred fromwaste stream chromatograms. Embodiments of the invention can beprogrammed to shut down a separation run or trigger an alarm conditionif the quality of collected samples falls outside of predeterminedlimits. The waste stream detector may also be used to direct thecollection of components, not collected in the primary fractioncollector, to a waste stream fraction collector for possible furtherpurification and tests.

Although the invention is described herein in terms of measuring timedifferences between detection of components in an eluent stream, personsskilled in the art should appreciate that flow volumes can also bemeasured and used to calibrate fraction collector timing signals withoutdeparting from the spirit and scope of the present invention.

Although the invention is described hereinbefore with respect toillustrative embodiments thereof, persons having ordinary skill in theart should appreciate that the foregoing and various other changes,omissions and additions in the form and detail thereof may be madewithout departing from the spirit and scope of the invention.

1-11. (canceled)
 12. A liquid chromatography apparatus comprising: aneluent stream flowing from a liquid chromatography column; a triggeringdetector operatively disposed in said eluent stream; said triggeringdetector configured to initiate a fraction collector timer upondetection by said triggering detector of a particular component in saideluent stream; a fraction collector operatively disposed in said eluentstream and operable in response to said fraction collector timer; and awaste stream flowing away from said fraction collector when saidfraction collector is closed; a waste stream detector operativelydisposed in said waste stream; said waste stream detector configured toacquire a waste stream chromatogram of said particular component whensaid component flows through said waste stream and providing timinginformation to calibrate the fraction collector delay time.
 13. Theapparatus according to claim 12 further comprising: a data systemconfigured to determine a fraction collector timing error according tocharacteristics of peaks in said waste stream chromatogram and adjustsaid fraction collector timer to reduce said timing error.
 14. Theapparatus according to claim 12 wherein said triggering detectorcomprises a U.V. detector.
 15. The apparatus according to claim 12wherein said triggering detector comprises a mass spectrometeroperatively disposed in an eluent stream branch that does not flow tosaid fraction collector.
 16. The apparatus according to claim 12 whereinsaid waste stream detector comprises a detector selected from the groupconsisting of a UV detector and a mass spectrometer.
 17. The apparatusaccording to claim 13 configured to calculate fraction collector timingerror by measuring the closed system arrival time (t₁) of a desiredcomponent at said waste stream detector when said fraction collector isheld closed; and comparing said closed system arrival time with thedetection time of and an edge of a remnant peak (t₂) by said wastestream detector.
 18. The apparatus according to claim 12 furthercomprising: means for computing timing error by comparing an arrivaltime of an edge of a remnant peak (t₂) detected in said waste streamwhen said fraction collector has collected a partial peak with anarrival time (t₁) of a peak detected in said waste stream when saidfraction collector is held closed.
 19. The apparatus according to claim12 further comprising means for confirming collection of said desiredcomponent by said fraction collector by automatic analysis of said wastestream chromatogram.
 20. The apparatus according to claim 12 whereinsaid triggering detector and said waste stream detector comprise asingle multi-channel instrument.